Etude de la colibactine dans les infections urinaires

Urinary tract infections (UTIs) are among the most common community-acquired or nosocomial bacterial infections. They are most often caused by uropathogenic Escherichia coli (UPECs). We assembled a collection of UPECs from 223 patients suffering from community-acquired UTIs in order to understand the role and impact of potential virulence factors of UPECs that have been little studied until now. In particular, we showed that 43% of UPECs carried a pathogenicity island, the pks island. It encodes a machinery for the synthesis of various secondary metabolites including a genotoxin, colibactin. In commensal intestinal E. coli, colibactin is suspected of promoting colorectal cancer. My aim was to study the impact and role of the pks island and its metabolites in the pathophysiology of UTIs. We have shown that the pks biosynthesis machinery is active in humans during UTI. For the first time, we found a marker metabolite for colibactin production in more than half of the urine of patients with pks+ UPEC UTIs. These clinical isolates of pks+ UPECs are genotoxic in vitro. By reproducing UTI in a mouse model, we demonstrated the expression of the pks machinery in intracellular bacterial communities formed in bladder cells and demonstrated the presence of DNA damage in urothelial cells related to colibactin production. This damage occurs even in KRT14+ progenitor cells of the bladder. This study suggests that UTIs may have longer-term consequences for the host, particularly in bladder cancer. In perspective, it seems also appropriate to question the current recommendation of no treatment for asymptomatic bacteriuria, which can persist for months or even years even though they are caused by strains that frequently carry the pks island and are genotoxic.Les infections urinaires (UTI) sont parmi les infections bactériennes communautaires ou nosocomiales les plus fréquentes. Elles sont le plus souvent causées par des Escherichia coli uropathogènes (UPEC). Nous avons constitué une collection d’UPEC issus de 223 patients souffrants d’UTI communautaires afin de comprendre le rôle et l’impact de potentiels facteurs de virulence des UPEC jusque-là peu étudiés. En particulier nous avons montré que 43% des UPEC portaient un îlot de pathogénicité, l’îlot pks. Il code pour une machinerie de synthèse de différents métabolites secondaires dont une génotoxine, la colibactine. Chez les E. coli commensales intestinales, la colibactine est suspectée de promouvoir le cancer colorectal. Mon objectif a été d’étudier l’impact et le rôle de l’îlot pks et de ses métabolites dans la physiopathologie des UTI. Nous avons montré que la machinerie de biosynthèse pks était active chez l’Homme durant l’UTI. Nous avons ainsi retrouvé pour la première fois un métabolite témoin de la production de la colibactine dans plus de la moitié des urines des patients atteints d’une UTI à UPEC pks+. Ces isolats cliniques d’UPEC pks+ sont génotoxiques in vitro. En reproduisant une UTI en modèle murin, nous avons démontré l’expression de la machinerie pks dans les communautés bactériennes intracellulaires formées dans les cellules vésicales et nous avons mis en évidence la présence de dommages à l’ADN dans les cellules urothéliales liés à la production de colibactine. Ces dommages surviennent y compris dans des cellules KRT14+ progénitrices de la vessie. Cette étude suggère que les UTI pourraient par ce biais avoir des conséquences à plus long terme sur l’hôte, en particulier dans le cancer de la vessie. En perspective, il convient également de questionner l'actuelle recommandation d'absence de traitement des bactériuries asymptomatiques, qui peuvent ainsi persister des mois voire des années alors qu’elles sont causées par des souches fréquemment porteuses de l’îlot pks et génotoxiques

Study of colibactin in urinary tract infections

Les infections urinaires (UTI) sont parmi les infections bactériennes communautaires ou nosocomiales les plus fréquentes. Elles sont le plus souvent causées par des Escherichia coli uropathogènes (UPEC). Nous avons constitué une collection d’UPEC issus de 223 patients souffrants d’UTI communautaires afin de comprendre le rôle et l’impact de potentiels facteurs de virulence des UPEC jusque-là peu étudiés. En particulier nous avons montré que 43% des UPEC portaient un îlot de pathogénicité, l’îlot pks. Il code pour une machinerie de synthèse de différents métabolites secondaires dont une génotoxine, la colibactine. Chez les E. coli commensales intestinales, la colibactine est suspectée de promouvoir le cancer colorectal. Mon objectif a été d’étudier l’impact et le rôle de l’îlot pks et de ses métabolites dans la physiopathologie des UTI. Nous avons montré que la machinerie de biosynthèse pks était active chez l’Homme durant l’UTI. Nous avons ainsi retrouvé pour la première fois un métabolite témoin de la production de la colibactine dans plus de la moitié des urines des patients atteints d’une UTI à UPEC pks+. Ces isolats cliniques d’UPEC pks+ sont génotoxiques in vitro. En reproduisant une UTI en modèle murin, nous avons démontré l’expression de la machinerie pks dans les communautés bactériennes intracellulaires formées dans les cellules vésicales et nous avons mis en évidence la présence de dommages à l’ADN dans les cellules urothéliales liés à la production de colibactine. Ces dommages surviennent y compris dans des cellules KRT14+ progénitrices de la vessie. Cette étude suggère que les UTI pourraient par ce biais avoir des conséquences à plus long terme sur l’hôte, en particulier dans le cancer de la vessie. En perspective, il convient également de questionner l'actuelle recommandation d'absence de traitement des bactériuries asymptomatiques, qui peuvent ainsi persister des mois voire des années alors qu’elles sont causées par des souches fréquemment porteuses de l’îlot pks et génotoxiques.Urinary tract infections (UTIs) are among the most common community-acquired or nosocomial bacterial infections. They are most often caused by uropathogenic Escherichia coli (UPECs). We assembled a collection of UPECs from 223 patients suffering from community-acquired UTIs in order to understand the role and impact of potential virulence factors of UPECs that have been little studied until now. In particular, we showed that 43% of UPECs carried a pathogenicity island, the pks island. It encodes a machinery for the synthesis of various secondary metabolites including a genotoxin, colibactin. In commensal intestinal E. coli, colibactin is suspected of promoting colorectal cancer. My aim was to study the impact and role of the pks island and its metabolites in the pathophysiology of UTIs. We have shown that the pks biosynthesis machinery is active in humans during UTI. For the first time, we found a marker metabolite for colibactin production in more than half of the urine of patients with pks+ UPEC UTIs. These clinical isolates of pks+ UPECs are genotoxic in vitro. By reproducing UTI in a mouse model, we demonstrated the expression of the pks machinery in intracellular bacterial communities formed in bladder cells and demonstrated the presence of DNA damage in urothelial cells related to colibactin production. This damage occurs even in KRT14+ progenitor cells of the bladder. This study suggests that UTIs may have longer-term consequences for the host, particularly in bladder cancer. In perspective, it seems also appropriate to question the current recommendation of no treatment for asymptomatic bacteriuria, which can persist for months or even years even though they are caused by strains that frequently carry the pks island and are genotoxic

The synergistic triad between microcin, colibactin, and salmochelin gene clusters in uropathogenic<em> Escherichia coli</em>

International audienceA functional synergy was previously demonstrated between microcin, salmochelin and colibactin islands in Escherichia coli strains from B2 phylogroup. We aimed to determine this association prevalence in uropathogenic E. coli, and whether it was predictive of the infection severity in a collection of 225 E. coli strains from urinary samples. The high prevalence of this triad, even if it wasn’t correlated with infection severity, suggested that it might not be a virulence factor per se within the urinary tract, but would promote its colonization. This triad would enable the strain to dominate the rectal reservoir with a minimal genetic cost

Screening for β-lactam resistance by penicillin G in the Streptococcus anginosus group challenged by rare strains with altered PBPs

International audienceAbstract Background Streptococcus anginosus group (SAG) strains are common pathogens causing abscesses and bacteraemia. They are generally susceptible to β-lactams, which constitute first-line treatment. EUCAST recommends testing penicillin G susceptibility to screen for β-lactam resistance. Isolates categorized as susceptible (negative screening) can be reported as susceptible to aminopenicillins and third-generation cephalosporins. Objectives To assess the reliability of penicillin G resistance screening in predicting β-lactam resistance in SAG blood culture isolates, and to investigate isolates for which this test would be unreliable. Methods We determined the susceptibility to penicillin G, amoxicillin and ceftriaxone of 90 SAG blood culture isolates, all with negative penicillin G resistance screening. β-Lactam-resistant strains were sequenced and compared with susceptible reference SAG strains. Results We detected two isolates displaying β-lactam resistance, especially to third-generation cephalosporins, despite negative screening for penicillin G resistance. For these isolates, amino acid substitutions were identified next to the essential PBP motifs SxxK, SxN and/or KS/TGS/T. Changes in these motifs have been previously linked to β-lactam resistance in Streptococcus pneumoniae. Conclusions Our study suggests that aminopenicillin and third-generation cephalosporin susceptibility should be determined for SAG strains in the event of severe infection as screening for penicillin G resistance might not be sufficient to detect resistance mechanisms that predominantly affect cephalosporins. The PBP sequencing of resistant SAG strains allowed us to detect amino acid changes potentially linked to β-lactam resistance

The Polyphosphate Kinase of Escherichia coli Is Required for Full Production of the Genotoxin Colibactin

International audienceColibactin induces DNA damage in mammalian cells and has been linked to the virulence of Escherichia coli and the promotion of colorectal cancer (CRC). By looking for mutants attenuated in the promoter activity of clbB encoding one of the key enzymes for the production of colibactin, we found that a mutant of the gene coding for the polyphosphate kinase (PPK) produced less colibactin than the parental strain. We observed this phenotype in different strains ranging from pathogens responsible for meningitis, urinary tract infection, or mouse colon carcinogenesis to the probiotic Nissle 1917. We confirmed the role of PPK by using an inhibitor of PPK enzymatic activity, mesalamine (also known as 5-aminosalicylic acid). Interestingly, mesalamine has a local anti-inflammatory effect on the epithelial cells of the colon and is used to treat inflammatory bowel disease (IBD). Upon treatment with mesalamine, a decreased genotoxicity of colibactin-producing E. coli was observed both on epithelial cells and directly on purified DNA. This demonstrates the direct effect of mesalamine on bacteria independently from its anti-inflammatory effect on eukaryotic cells. Our results suggest that the mechanisms of action of mesalamine in treating IBD and preventing CRC could also lie in the inhibition of colibactin production. All in all, we demonstrate that PPK is required for the promoter activity of clbB and the production of colibactin, which suggests that PPK is a promising target for the development of anticolibactin and antivirulence strategies.IMPORTANCE Colibactin-producing E. coli induces DNA damage in eukaryotic cells and promotes tumor formation in mouse models of intestinal inflammation. Recent studies have provided strong evidence supporting the causative role of colibactin in human colorectal cancer (CRC) progression. Therefore, it is important to understand the regulation of the production of this genotoxin. Here, we demonstrate that polyphosphate kinase (PPK) is required for the promoter activity of clbB and the production of colibactin. Interestingly, PPK is a multifunctional player in bacterial virulence and stress responses and has been proposed as a new target for developing antimicrobial medicine. We observed inhibition of colibactin production by using a previously identified PPK inhibitor (i.e., mesalamine, an anti-inflammatory drug commonly prescribed for inflammatory bowel diseases). These data brought us a new perspective on the regulatory network of colibactin production and provided us a clue for the development of anticolibactin strategies for CRC treatment/prophylaxis

Oxygen concentration modulates colibactin production

International audienceUp to 25% of the E. coli strains isolated from the feces of healthy humans harbor the pks genomic island encoding the synthesis of colibactin, a genotoxic metabolite. Evidence is accumulating for an etiologic role of colibactin in colorectal cancer. Little is known about the conditions of expression of colibactin in the gut. The intestine is characterized by a unique oxygenation profile, with a steep gradient between the physiological hypoxic epithelial surface and the anaerobic lumen, which favors the dominance of obligate anaerobes. Here, we report that colibactin production is maximal under anoxic conditions and decreases with increased oxygen concentration. We show that the aerobic respiration control (ArcA) positively regulates colibactin production and genotoxicity of pks+ E. coli in response to oxygen availability. Thus, colibactin synthesis is inhibited by oxygen, indicating that the pks biosynthetic pathway is adapted to the anoxic intestinal lumen and to the hypoxic infected or tumor tissue

The Polyamine Spermidine Modulates the Production of the Bacterial Genotoxin Colibactin

Colibactin is a polyketide/nonribosomal peptide produced by Escherichia coli strains that harbor the pks island. This toxin induces DNA double-strand breaks and DNA interstrand cross-links in infected eukaryotic cells. Colibactin-producing strains are found associated with colorectal cancer biopsy specimens and promote intestinal tumor progression in various murine models. Polyamines are small polycationic molecules produced by both microorganisms and eukaryotic cells. Their levels are increased in malignancies, where they contribute to disease progression and metastasis. In this study, we demonstrated that the endogenous spermidine synthase SpeE is required for full genotoxic activity of colibactin-producing E. coli. Supplying spermidine in a ΔspeE pks+ E. coli strain restored genotoxic activity. Spermidine is involved in the autotoxicity linked to colibactin and is required for direct damaging activity on DNA. The production of the colibactin prodrug motif is impaired in ΔspeE mutants. Therefore, we demonstrated that spermidine has a direct impact on colibactin synthesis

A Toxic Friend: Genotoxic and Mutagenic Activity of the Probiotic Strain Escherichia coli Nissle 1917

International audienceThe probiotic Escherichia coli strain Nissle 1917 (DSM 6601, Mutaflor), generally considered beneficial and safe, has been used for a century to treat various intestinal diseases. However, Nissle 1917 hosts in its genome the pks pathogenicity island that codes for the biosynthesis of the genotoxin colibactin. Colibactin is a potent DNA alkylator, suspected to play a role in colorectal cancer development. We show in this study that Nissle 1917 is functionally capable of producing colibactin and inducing interstrand cross-links in the genomic DNA of epithelial cells exposed to the probiotic. This toxicity was even exacerbated with lower doses of the probiotic, when the exposed cells started to divide again but exhibited aberrant anaphases and increased gene mutation frequency. DNA damage was confirmed in vivo in mouse models of intestinal colonization, demonstrating that Nissle 1917 produces the genotoxin in the gut lumen. Although it is possible that daily treatment of adult humans with their microbiota does not produce the same effects, administration of Nissle 1917 as a probiotic or as a chassis to deliver therapeutics might exert long-term adverse effects and thus should be considered in a risk-versus-benefit evaluation. IMPORTANCE Nissle 1917 is sold as a probiotic and considered safe even though it has been known since 2006 that it harbors the genes for colibactin synthesis. Colibactin is a potent genotoxin that is now linked to causative mutations found in human colorectal cancer. Many papers concerning the use of this strain in clinical applications ignore or elude this fact or misleadingly suggest that Nissle 1917 does not induce DNA damage. Here, we demonstrate that Nissle 1917 produces colibactin in vitro and in vivo and induces mutagenic DNA damage. This is a serious safety concern that must not be ignored in the interests of patients, the general public, health care professionals, and ethical probiotic manufacturers

Deciphering the interplay between the genotoxic and probiotic activities of Escherichia coli Nissle 1917

International audienceAlthough Escherichia coli Nissle 1917 (EcN) has been used therapeutically for over a century, the determinants of its probiotic properties remain elusive. EcN produces two siderophore-microcins (Mcc) responsible for an antagonistic activity against other Enterobacteriaceae. EcN also synthesizes the genotoxin colibactin encoded by the pks island. Colibactin is a virulence factor and a putative pro-carcinogenic compound. Therefore, we aimed to decouple the antagonistic activity of EcN from its genotoxic activity. We demonstrated that the pks-encoded ClbP, the peptidase that activates colibactin, is required for the antagonistic activity of EcN. The analysis of a series of ClbP mutants revealed that this activity is linked to the transmembrane helices of ClbP and not the periplasmic peptidase domain, indicating the transmembrane domain is involved in some aspect of Mcc biosynthesis or secretion. A single amino acid substitution in ClbP inactivates the genotoxic activity but maintains the antagonistic activity. In an in vivo salmonellosis model, this point mutant reduced the clinical signs and the fecal shedding of Salmonella similarly to the wild type strain, whereas the clbP deletion mutant could neither protect nor outcompete the pathogen. The ClbP-dependent antibacterial effect was also observed in vitro with other E. coli strains that carry both a truncated form of the Mcc gene cluster and the pks island. In such strains, siderophore-Mcc synthesis also required the glucosyltransferase IroB involved in salmochelin production. This interplay between colibactin, salmochelin, and siderophore-Mcc biosynthetic pathways suggests that these genomic islands were co-selected and played a role in the evolution of E. coli from phylogroup B2. This co-evolution observed in EcN illustrates the fine margin between pathogenicity and probiotic activity, and the need to address both the effectiveness and safety of probiotics. Decoupling the antagonistic from the genotoxic activity by specifically inactivating ClbP peptidase domain opens the way to the safe use of EcN

A Toxic Friend: Genotoxic and Mutagenic Activity of the Probiotic Strain Escherichia coli Nissle 1917

International audienceThe probiotic Escherichia coli strain Nissle 1917 (DSM 6601, Mutaflor), generally considered beneficial and safe, has been used for a century to treat various intestinal diseases. However, Nissle 1917 hosts in its genome the pks pathogenicity island that codes for the biosynthesis of the genotoxin colibactin. Colibactin is a potent DNA alkylator, suspected to play a role in colorectal cancer development. We show in this study that Nissle 1917 is functionally capable of producing colibactin and inducing interstrand cross-links in the genomic DNA of epithelial cells exposed to the probiotic. This toxicity was even exacerbated with lower doses of the probiotic, when the exposed cells started to divide again but exhibited aberrant anaphases and increased gene mutation frequency. DNA damage was confirmed in vivo in mouse models of intestinal colonization, demonstrating that Nissle 1917 produces the genotoxin in the gut lumen. Although it is possible that daily treatment of adult humans with their microbiota does not produce the same effects, administration of Nissle 1917 as a probiotic or as a chassis to deliver therapeutics might exert long-term adverse effects and thus should be considered in a risk-versus-benefit evaluation. IMPORTANCE Nissle 1917 is sold as a probiotic and considered safe even though it has been known since 2006 that it harbors the genes for colibactin synthesis. Colibactin is a potent genotoxin that is now linked to causative mutations found in human colorectal cancer. Many papers concerning the use of this strain in clinical applications ignore or elude this fact or misleadingly suggest that Nissle 1917 does not induce DNA damage. Here, we demonstrate that Nissle 1917 produces colibactin in vitro and in vivo and induces mutagenic DNA damage. This is a serious safety concern that must not be ignored in the interests of patients, the general public, health care professionals, and ethical probiotic manufacturers